1. Field of the Invention
The present invention relates to the field of DNA sequencing and to the fields of calorimetry and potentiometry for chemical analysis.
2. Related Art
The essence of biology is a deep understanding of all of the species and their biological mechanisms. Speciation and biological function are primarily determined by the organism's DNA sequence. The development of vastly improved DNA sequence determination for personalized medicine and ecological studies could complete the revolution initiated by the Human Genome Project. The Human Genome Project was essentially accomplished by a reduction in the cost of DNA sequencing by three orders of magnitude. It is desired to reduce the cost by another three orders of magnitude to enable profiling of individuals genome. To achieve this goal, a highly integrated platform will be needed.
Current sequencing technologies involve a method of DNA sequencing known as sequencing by synthesis (SBS). See, for example, Seo et al. “Four-color DNA sequencing by synthesis on a chip using photocleavable fluorescent nucleotides,” PNAS 102: 5926-5959 (Apr. 26, 2005). As described there, SBS was first introduced around 1988, See Hyman, “New method of sequencing DNA,” Anal. Biochem., 174: 423-436, (1 Nov. 1988). The method works by measuring pyrophosphate generated by the DNA polymerization reaction. DNA and DNA polymerase are held by a DEAE-Sepharose column and solutions containing different dNTPs are pumped through. The pyrophosphate generated is measured continuously by a device consisting of a series of columns containing enzymes covalently attached to Sepharose.
One approach to sequencing by detecting pyrophosphate is the pyrosequencing method, which is being commercialized by Biotage and 454 Life Sciences (a subsidiary of CuraGen Corp., Branford, Conn.).
Pyrosequencing is based on real-time bioluminometric detection of released pyrophosphate as a result of successful nucleotide incorporation. The released pyrophosphate is converted to ATP-by-ATP sulfurylase and the level of ATP is sensed by a luciferase producing proportional light signal, which is detected by photosensing devices. Biotage is performing this assay in 96 well format and 454 Life Sciences perform the reaction in picotiter plate format for analysis of more than 100,000 DNA fragments simultaneously.
Pyrosequencing is further described in Ronaghi, M., Uhlen, M., and Nyren, P. 1998, Science 281: 363, “A sequencing method based on real-time pyrophosphate.” 454's technology is based on performing hundreds of thousands of simultaneous sequencing reactions in small volume wells on plates. All molecular biology reactions—DNA amplification, sequencing by synthesis, and signal light generation—occur in a single well.
An extension of the original “fluorescent in situ sequencing,” termed bead-based polony sequencing, was developed by Jay Shendure and colleagues in George Church's Lab at the Lipper Center for Computational Genetics, Harvard Medical School, Boston. In this sequencing-by-synthesis approach, short fragment DNA libraries are clonally amplified onto 1-μm beads and embedded into a polymer matrix on the surface of microscope slides. The polony slides are then placed into an automated flow cell, where four-color, fluorescently labeled reagents (corresponding to the DNA bases, A, C, G, or T) are delivered to serially sequenced DNA strands.
Other DNA sequencing methods have been proposed. One approach to generating paired genome-fragment tags uses an emulsion PCR-based amplification step, an optimized polymerase colony (polony)-based sequencing-by-ligation protocol and a conventional epifluorescence microscope with a sophisticated algorithm that allows researchers to stitch together the fragmented sequence reads into one continuous thread. See Science 309, 1728-1732, 2005.
Specific Patents and Publications
Wang et al., “Continuous Flow Thermal Cycler Microchip for DNA Cycle Sequencing,” Anal. Chem., 78 (17), 6223-6231, 2006, discloses a polymer-based continuous flow thermal cycler (CFTC) microchip for Sanger cycle sequencing using dye terminator chemistry. The CFTC chip consisted of a 20-loop spiral microfluidic channel hot-embossed into polycarbonate (PC) that had three well-defined temperature zones poised at 95, 55, and 60° C. for denaturation, renaturation, and DNA extension, respectively.
U.S. Pat. No. 5,149,625 to Church, et al., issued Sep. 22, 1992, entitled “Multiplex analysis of DNA,” discloses a method including the steps of: a) ligating the DNA into a vector comprising a tag sequence, the tag sequence includes at least 15 bases, wherein the tag sequence will not hybridize to the DNA under stringent hybridization conditions and is unique in the vector, to form a hybrid vector, b) treating the hybrid vector in a plurality of vessels to produce fragments comprising the tag sequence, wherein the fragments differ in length and terminate at a fixed known base or bases, wherein the fixed known base or bases differs in each vessel, c) separating the fragments from each vessel according to their size, d) hybridizing the fragments with an oligonucleotide able to hybridize specifically with the tag sequence, and e) detecting the pattern of hybridization of the tag sequence, wherein the pattern reflects the nucleotide sequence of the DNA.
U.S. Pat. No. 4,863,849 to Melamede, issued Sep. 5, 1989, entitled “Automatable process for sequencing nucleotide,” discloses a sequencing by synthesis method which involves adding an activated nucleotide precursor (a nucleoside 5′-triphosphate) having a known nitrogenous base to a reaction mixture comprising a primed single-stranded nucleotide template to be sequenced and a template-directed polymerase. The reaction conditions are adjusted to allow incorporation of the nucleotide precursor only if it is complementary to the single-stranded template at the site located one nucleotide residue beyond the 3′ terminus of the primer. After allowing sufficient time for the reaction to occur, the reaction mixture is washed so that unincorporated precursors are removed while the primed template and polymerase are retained in the reaction mixture.
U.S. Pat. No. 5,302,509 to Cheeseman, issued Apr. 12, 1994, entitled “Method for sequencing polynucleotides,” discloses a method for determining the sequence of nucleotides on a single strand DNA molecule. The single strand DNA molecule is attached to a leader oligonucleotide and its complementary strand to a solid-state support. Fluorescently labeled 3′-blocked nucleotide triphosphates, with each of the bases A, G, C, T having a different fluorescent label, are mixed with the bound DNA molecule in the presence of DNA polymerase.
US 2004/0142330 to Nyren, et al., published Jul. 22, 2004, entitled “Method of sequencing DNA,” discloses a method of pyrosequencing which use an α-thio analogue of deoxy ATP (dATP) (or dideoxy ATP (ddATP)) namely an (1-thio) triphosphate (or α-thiophosphate) analogue of deoxy or dideoxy ATP, preferably deoxyadenosine[1-thio]triphosphate. Use of these modified analogues is an improvement to the basic PPi-based sequencing method in which one uses in place of dATP, a dATP analogue (specifically dATP α-s) which is incapable of acting as a substrate for luciferase, but which is nonetheless capable of being incorporated into a nucleotide chain by a polymerase enzyme (WO98/13523).
Further improvements to the basic PPi-based sequencing technique include the use of a nucleotide degrading enzyme such as apyrase during the polymerase step, so that unincorporated nucleotides are degraded, as described in WO 98/28440, and the use of a single-stranded nucleic acid binding protein in the reaction mixture after annealing of the primers to the template, which has been found to have a beneficial effect in reducing the number of false signals, as described in WO00/43540.
US 2003/0082583 by Hassibi, et al., published May 1, 2003, entitled “Bioluminescence regenerative cycle (BRC) for nucleic acid quantification,” discloses another technique that employs pyrophosphate. In BRC, steady state levels of bioluminescence result from processes that produce pyrophosphate. Pyrophosphate reacts with APS in the presence of ATP sulfurylase to produce ATP. The ATP reacts with luciferin in a luciferase-catalyzed reaction, producing light and regenerating pyrophosphate. The pyrophosphate is recycled to produce ATP and the regenerative cycle continues.
Another, different, reaction sensor is disclosed in U.S. Pat. No. 6,638,716 to Heller, et al., issued Oct. 28, 2003, entitled “Rapid amperometric verification of PCR amplification of DNA.” This device utilizes an electrode coated with a redox polymer film. The redox polymer film is preferably a redox hydrogel. A binding agent is immobilized in the redox polymer film, preferably through covalent bonding of the binding agent to the redox polymer. The DNA is labeled, while amplified, with two or more different ligands, the first of which binds strongly to the binding agent immobilized in the redox polymer film. When the sample in which the amplification is to be confirmed is contacted with the electrode, amplified DNA is immobilized on the electrode through linkage of the immobilized binding agent in the redox polymer film with the first ligand. The presence of the amplified DNA on the electrode is detected through exposure of the electrode to a detection marker. The detection marker is a molecule with two functional groups. One of the functional groups binds with the second ligand of the amplified DNA; the second functional group of the detection marker produces an electrochemically detectable signal.